3-(aminoacyl-amino)-saccharides and processes for their preparation

ABSTRACT

The invention pertains to 3-(aminoacyl-amino)-saccharides of the general formula (I), ##STR1## wherein R&#39; is selected from the group consisting of hydrogen, a carboxyl group, a phenyl group and an alkyl group with 1-10 C-atoms which is optionally substituted by a phenyl, carboxyl, hydroxyl, mercapto or amino group, wherein said substitutents are optionally protected with protective groups; R 2  is selected from the group consisting of hydrogen, an amino protective group and a peptide group; and R 3  is hydrogen or a fructosyl radical; and n is 0 or 1. The invention also pertains to a method for preparing them.

The invention concerns 3-(aminoacyl-amino)-saccharides and processes fortheir preparation from 3-amino-allo-saccharose.

Linkings of amino acids and saccharides are mainly present inglycopeptides and glycoproteins and, to a small extent, also inbacterial lipopolysaccharides. In the case of glycopeptides, it is aquestion of partial structures which occur as linkage region ofsaccharides and proteins in glycoproteins. Glycoproteins, which are tobe found in soluble form in the blood and in numerous secrets, as wellas in fixed form in membrane double layers, have, in recent times,achieved interest since their function has been recognised andinvestigated in biological control processes. In many cases, thecarbohydrate side chains serve as recognition signal.

The nature of the covalent bonding between protein and carbohydrate sidechain scarcely differs, however, in spite of the great number ofnaturally-occurring glycoproteins, which is caused by the biosynthesisof the glycoproteins. The two components are linked via a glycosidicbond, whereby one differentiates between N-glycoproteins andO-glycoproteins. In the case of the N-glycoproteins, the side chainamide group of an asparagine component is mostly linkedβ-N-glycosidically with 2-acetamido-2-desoxy-D-glucose. However, inaddition, in recent times, there have also been found N-glycosyl sudN-galsctosyl structures. In the case of O-glycoproteins, there is mostlypresent an α-O-glycosidic linking of 2-acetamido-2-desoxy-D-galactose ora β-O-glycosidic linkage of D-xylose with a hydroxyl group of serine orthreonine (H. Paulsen, Synthesen, Konformationen undRotgen-struktureanalysen von Saccharidketteh der Core-Regionen vonGlycoproteinen, Angew. Chem. 102 (1990) 851-867; H. Kurtz, Synthese vonGlycopeptiden - Partialstrukturen biologischer Erkennungskomponenten,Angew. Chem. 99 (1987) 297-311; J. Montreuil, Primary Structure ofGlycoprotein Glycans, Adv. Carbohydr. Chem. Biochem. 37 (1980) 157-223).

Individual amino acids have also been detected in lipopolysaccharides ofbacterial origin which are linked via an amide formation withaminosugars, e.g. N-acetyl-glycine, which is bound via the amino groupof 4-amino-4,6-didesoxy-D-glycopyranosyl radical to the O-specific sidechain of the lipopolysaccharide of Shigella dysenteriae type 7 (Y. A.Knirel et al., Carbohydr. Res. 179 (1988), 51-60), or N-acetyl-L-serinewhich is bound via the amino group of the3-amino-3,6-didesoxy-D-glucopyranosyl radical with the O-specific sidechain of the lipopolysaccharide of Escherichia coli 0114 (V. L. L'vov etal., Carbohydr. Res. 112 (1983) 233-239). These substances actantigenically.

The invention refers to new 3-(aminoacyl-amino)-saccharides of thefollowing formula I, ##STR2## in which R¹ signifies hydrogen, a carboxylgroup or a phenyl group or an alkyl group with 1-10 C-atoms which ispossibly substituted by a phenyl, carboxyl, hydrozyl, mercapto or aminogroup, whereby the said substituents are possibly provided withprotective groups, R² is hydrogen, an amino protective group usual inpeptide chemistry or a peptide group and R³ represents hydrogen or afructosyl group, n=0 or 1, especially 3-(L-aminoacyl-amino)-D-allopyranosyl-β-D-fructofuranoside in thefollowing designated as 3-(aminoacyl-amino)-allo-saccharose, and3-(L-aminoacyl-amino)-D-allopyranose. Furthermore, the inventionincludes preparation processes for these materials starting from3-amino-3-desoxy-D-allo-pyranosyl-β-D-fructofuranoside, briefly called3-amino-allo-saccharose.

In the case of the 3-(aminoacyl-amino)-allo-saccharides according to theinvention, it is a question of materials in which the carboxyl group ofan amino acid is bound via an acid amide binding to the amino group ofthe 3-amino-allo-saccharose. Thus, in contra-distinction to theglycoproteins, these compounds contain no glycosidic bond betweencarbohydrate and amino acid but rather show a similar bonding structureto the above-mentioned lipopolysaccharides.

The 3-(aminoacyl-amino)-allo-saccharoses makes possible, with help ofthe methods of glycopeptide synthesis, the preparation of novelglycopeptides in which carbohydrate and protein are not bound via anN-glycosidic bond but rather via an acid amide bond. As startingcompounds herefor are suitable especially acidic and basic amino acidssince the amino acid residues in the amino acid saccharides in each casecontain two functional groups, thus the peptide chain can be built up ontwo sides.

On the basis of the polyfunctionality of both starting components, thepreparation of glycopeptides is very problematical and mostly requiresthe use of protective groups not only on the saccharide but also on theamino acid or the peptide. By the use of the 3-amino-allo-saccharose,which can be obtained without introduction of protective groups, it isnow possible completely to omit protective groups for the synthesis ofthe 3-aminoacyl group in the saccharide part.

Via the splitting off of the fructosyl radical of the3-(aminoacyl-amino)-allo-saccharoses, one can now also get to3-(aminoacyl-amino)-alloses which, for their part, can serve for thepreparation of glycopeptides. In this case, not only can a peptide chainbe built up, in that further amino acids are attached to the amino acidradical, but also an oligosaccharide chain can be produced via theallose,

The compounds according to the invention, as well as derivativesproduced therefrom, are useable in the interdisciplinary research anduse, e.g. for the elucidation of the biological function ofglycoproteins, for the induction of antibodies, for the research of thebiological recognition and selectivity and for the development ofpharmaceutically active materials, as well as as potential inhibitor forenzymes and micro-organisms.

The preparation of the 3-(L-aminoacyl)-amino-allo-saccharides startsfrom saccharose. Into this poly-functional molecule is introduced a ketogroup on the C-3 of the glucosyl radical, by means of a microbialoxidation with Agrobacterium tumefaciens, which subsequently can beselectively converted into an amino group by means of reductiveamination, e.g. with ammonia, hydroxylamine or hydrazine by means ofhydrogen and metal catalyst. After chromatographic purification, oneobtains the amination product 3-amino-allo-saccharose (M. Pietsch,Dissertation TU Braunschweig, 1993). This synthesis route has alreadybeen described in similar way for the preparation of diamines fromreduced saccharides in EP 0 399 448 A2.

The 3-amino-allo-saccharose now serves, for its part, as startingproduct far the 3-(aminoacyl-amino)-allo-saccharides. Surprisingly, thereaction takes place following the process described by M. Kiyozumi etal., Carbohydr. Res. 14 (1970) 355-364. For this purpose, the3-amino-allo-saccharose is reacted with N-terminally protected amino anddiamino acids or with N- and C-terminally protected aminodicarboxylicacids or correspondingly protected peptides in a pyridine-water mixturewith activation of the carboxyl function of the amino acid by means ofN,N'-dicyclohexylcarbodiimide (DCC). Subsequently, the protective groupscan be split off. A purification of the reaction products is possiblee.g. by column chromatography. The structural verification of thepurified substances takes place by means of ¹³ C-NMR spectroscopy andfast atom bombardment mass spectrometry (FAB-MS).

As process for the preparation of the 3-(aminoacyl-amino)-saccharides,the following steps are taken:

1. Oxidation of saccharose e.g. with bacteria of the strainAgrobacterium tumefaciens NCPPB 396 to 3-keto-saccharose.

2. Reductive amination of the 3-keto-saccharose, as well as working upand separation of the resulting product mixture and isolation of the3-amino-allo-saccharose.

3. N-acylation of the 3-amino-allosaccharose with amino acids orpeptides with an activated carboxyl group, the other amino and carboxylgroups of which are possibly protected by means of protective groupsusual in peptide chemistry.

4. Splitting off of one or more protective groups from the amino acidradical of the substance and/or of the fructosyl radical from thesaccharose group, as well as isolation and purification of the product.

For the activation of the carboxyl group, dicyclo-hexylcarbodiimide(DCC) is preferred but 1,2-dihydro-2-ethoxyquinoline-1-carboxylic acidethyl ester (EEDQ) is also useable as coupling resgent and otheractivated carboxyl groups, such as anhydrides, esters, azides or halidesinsofar as they do not react to a greater extent with the hydroxylgroups of the saccharide.

As amino protective groups, all groups known from peptide chemistry areuseable which can again be split off in a gentle way. For example, theremay be mentioned benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (BOC) andtriphenylmethyl (Trt), which are again split off by catalytichydrogenation or acidic hydrolysis with hydrohalic acids but especiallywith trifluoroacetic acid.

As carboxyl protective groups are to mentioned, for example, alkylesters and especially benzyl esters, which can be split off acidicallyor alkaline. Furthermore, the benzyl groups are to be removed especiallygently by hydrogenolysis.

As amino acids are preferred the "natural" amino acids obtainable inlarge amounts by hydrolysis of proteins but, for certain purposes,synthetically prepared racemic or D-amino acids or amino acids which donot occur in nature can also be used. Furthermore, instead of an aminoacid, a corresponding peptide can also be used.

The invention is explained in more detail by the following Examples.

EXAMPLE 1 Coupling of 3-amino-allo-saccharose with Amino Acids whichContain Pure Hydrocarbon Side Chains 3-(L-leucyl-amino)-allo-saccharose

200 mg 3-amino-allo-saccharose, together with 137 mg N-BOC-L-leucine(0.059 mmol), are dissolved in 13 ml of solvent (pyridine/H₂ O, 4:1) andcooled in an icebath. Subsequently, with cooling, DCC (1.0 mmoldissolved in 0.5 ml solvent) is slowly added dropwise with stirring andcooling. After some time, the cooling is ended. After about 17 hoursreaction time (with stirring), the reaction is broken off by addition ofa drop of glacial acetic acid to the reaction mixture and, after 15minutes stirring, the resulting insoluble dicyclohexylurea is filteredoff. With the addition of toluene, the solvent is gently removed on arotary evaporator (40° C., vacuum). The reaction product is transferredwith water and diethyl ether into a separating funnel and the aqueousphase extracted several times with diethyl ether. The reaction productin the aqueous phase is freeze dried and the product purified by meansof column chromatography (stat. phase:silica gel, eluent: acetic acidethyl ester/ethanol/water, 5:3:1). One obtains3-(N-t-BOC-L-leucyl-amino)-allo-saccharose in 35% yield.

C₂₃ H₄₂ N₂ O₁₃ (M=554 g/mol)

¹³ C-NMR data (75.5 MHz, D₂ O/acetone-d₆)=178.0 (C1"), 158.3 (C7"),105.0 (C2'), 9.30 (C1), 82.6 (C5'), 82.2 (C8"), 77.5 (C3'), 74.6 (C4'),69.4/66.1/65.7 (C2,4,5), 62.9/62.4 (C6',1'), 60.8 (C6), 55.3/53.4(C3,2"), 40.8 (C3"), 28.5 (C9"-11"), 25.1 (C4"), 23.1 (C5",6").

FAB mass spectrum (pos. matrix glycerol):

m/z=555 [M+H]⁺, 393 [M-fructosyl radical+H]⁺, 761 [M=2 glycerol+Na]⁺.

The splitting off of the t-butyloxycarbonyl (BOC) protective group takesplace with 90% trifluoroacetic acid: For this purpose, the reactionproduct is dissolved in cold 90% trifluoroacetic acid and left to standfor 20 to 30 min at 4° C. Subsequently, the substance is precipitatedout by addition of cold dry diethyl ether, filtered off and after-washedseveral times with diethyl ether. After column chromstographicpurification, one obtains 3-(L-leucyl-amino)-allo-saccharose.

In the same way are prepared:

3-(N-BPC-L-alanyl-amino)-allo-saccharose C₂₀ H₃₆ N₂ O₁₃ 3

(M=512 g/mol)

¹³ C-NMR data (75.5 MHz, D₂ O/acetone-d₆):

δ=170.8 (C1"), 154.9 (C4"), 104.2 (C2'), 91.6 (C1), 82.7 (C5'), 78.2(C5"), 76.4 (C3'), 73.7 (C4'), 69.0/65.5/65.0 (C2,4,5), 61.8 (C6',1'),60.1 (C6), 55.3 (C3),

49.5 (C2"), 28.1 (C6"-8"), 17.9 (C2").

3-(N-BOC-L-phenylalanyl-amino)-allo-saccharose C₂₆ H₄₀ N₂ O₁₃

(M=588 g/mol)

¹³ C-NMR data (75.5 MHz, D₂ O/acetone-d₆ :

δ=176.3 (C1"), 158.0 (C10"), 137.0 (C4"), 128.9/128.5/128.1 (C5"-9"),104.5 (C2'), 92.5 (C1), 82.4 (C5'), 81.0 (C11"), 77.4 (C3'), 74.3 (C4'),69.2/65.7/65.4 (C2,4,5), 66.5 (C6'), 62.4 (C1'), 60.5 (C6), 56.0/53.0(C3,2"), 40.5 (C3"), 28.1 (C12"-14").

FAB mass spectrum (pos. matrix glycerol):

m/z=589 [M+H]⁺, 427 [M-fructosyl radical+H], 611 [M+Na]⁺.

The BOC group can also be split off from these compounds withtrifluoroacetic acid.

EXAMPLE 2 Coupling of 3-amino-allo-saccharose with Acid and Basic AminoAcids 3-(4-L-aspartyl-amino) -allo-saccharose C₁₆ H₂₈ N₂ O₁₃

For this purpose, 150 mg 3-amino-allo-saccharose (0.44 mol), togetherwith 142 mg N-t-BOC-L-aspartic acid benzyl ester, are dissolved in 13 mlof solvent (pyridine/H₂ O, 4:1) and cooled in an icebath. Subsequently,with cooling, DCC (0.7 mmol dissolved in 0.5 ml of solvent) is slowlyadded dropwise with stirring and cooling. After some time, the coolingis ended. After about 17 hours reaction time (with stirring), thereaction is broken off by addition of a drop of glacial acetic acid tothe reaction mixture and after 15 minutes stirring the resultantinsoluble dicyclohexylures is filtered off. With the addition oftoluene, the solvent is gently removed on a rotary evaporator (40° C.vacuum) The reaction product is transferred with water and diethyl etherinto a separating funnel and the aqueous phase extracted several timeswith diethyl ether. The reaction product in the aqueous phase is freezedried.

The splitting off of the benzyl protective group on the α-carboxyl groupof the aspartic acid radical takes place by hydrogenolysis. Pot thispurpose, the freeze-dried crude product (200 mg) is dissolved in 22 mlmethanol in a three-necked flask with reflux cooler. The solution istreated with through-flowing hydrogen in the presence of a catalyst(palladium/active carbon, 10% Pd) with stirring for 3 h at a temperatureof 30° to 40° C. after atmospheric oxygen had previously been displacedby nitrogen. After filtering off of the catalyst and removal of thesolvent on a rotary evaporator (30° C. vacuum) the product is purifiedby means of column chromatography (stat, phase:silica gel, eluent:aceticacid ethyl ester/ethanol/water, 5:3:1). One obtains3-(N-BOC-4-L-aspartyl-amino)-allo-sacaharose in 44% yield.

The splitting off of the BOC protective group takes place with 90%trifluoroscetic acid: for this purpose, the reaction product isdissolved in 0.7 ml cold 90% trifluoroscetic acid and left to stand for15 to 20 min at 4° C. Subsequently, the substance is precipitated out byaddition of cold dry diethyl ether, filtered off and after-washedseveral times with diethyl ether. After column chromatographicpurification, one obtains 3-(4-L-aspartyl-amino)-allo-saccharose (77%yield).

¹³ C-NMR data (75.5 MHz, D₂ O/acetone-d₆):

δ=174.7/174.1 (C1",4"), 104.8 (C2'), 93.2 (C1), 82.7 (C5'), 77.3 (C3'),74.9 (C4'), 69.6/66.2/65.9 (C2,4,5), 63.2/62.3 (C6',1'), 61.1 (C6),53.5/52.5 (C3,2"), 37.3 (C3").

FAB mass spectrum (pos. matrix glycerol):

m/z - 457 [M+H]⁺, 295 [M-fructosyl radical+H]⁺, 913 [2M+H]⁺

In the same way is prepared3-(Nα-BOC-N-CBZ-L-lysyl)-amino-allo-saccharose C₃₁ H₄₉ N₃ O₁₄ (M=703g/mol).

¹³ C-NMR date (75.5 MHz, D₂ O/acetone-d₆):

δ=176.3 (C1"), 158.0/157.9 (C7", 12"), 137.0 (C14"), 128.9/128.1/128.0(C15"-19"), 104.5 (C2'), 92.5 (C1), 82.4 (C5'), 81.0 (C8"), 77.4 (C3'),74.3 (C4'), 69.2/65.7/65.4 (C2,4,5), 66.5 (C6'), 62.4 (C1'), 60.5 (C6),56.1/53.0 (C3,2"), 40.5/40.6 (C3"-6", 13"), 28.1 (C9"-11").

FAB mass spectrum (pos. matrix glycerol):

m/z=726 [M+Na]⁺.

By splitting off of the protective groups with trifluoroacetic acid andcatalytic hydrogenation, one obtains therefrom3-(L-lysyl)-amino-allo-saccharose.

EXAMPLE 3 Hydrolyric Cleavage of the Glycoside Bond of theL-aspartyl-amino)-allo-saccharose

By acid hydrolysis, one obtains from the3-(4-L-aspartyl-amino)-allo-saccharose theD-(4-L-aspartyl-amino)-allose. For this purpose, 150 mg3-(4-L-aspartyl-amino)-allo-saccharose are heated for 10 min to 60° C.with 5 ml hydrochloric acid (0.5N). Subsequently, the reaction solutionis immediately cooled to 20° C. and neutralised with caustic sodasolution. The separating off of the product from the reaction mixturetakes place by means of preparative liquid chromatography on a cationexchanger in the Ca²⁺ form (eluent: H₂ O, 70° C).3-(4-L-aspartyl-amino)-allose is obtained with a yield of 80%, referredto the 3-(4-L-aspartyl-amino)-allo-saccharose used.

We claim:
 1. A 3-(aminoacyl-amino)-saccharide of the general formula I,##STR3## wherein R¹ is selected from the group consisting of hydrogen, acarboxyl group, a phenyl group and an alkyl group with 1-10 C-atomswhich is optionally substituted by a phenyl, carboxyl, hydroxyl,mercapto or amino group, wherein said substitutents are optionallyprotected with protective groups; R² is selected from the groupconsisting of hydrogen, an amino protective group and a peptide group;and R³ is hydrogen or a fructosyl radical; and n is 0 or
 1. 2. Asaccharide according to claim 1, selected from the group of compoundsconsistingof:3-(4-L-aspartyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside,3-(L-alanyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside,3-(L-leucyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside,3-(L-lysyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside,3-(L-phenylalanyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside,and 3-(4-L-aspartyl-amino)-3-desoxy-D-allopyranose.
 3. A process for thepreparation of a 3-(aminoacyl-amino)-saccharide according to claim 1,comprising reacting a3-amino-3-desoxy-D-allopyranosyl-β-D-fructofuranoside with a compound ofthe formula II, ##STR4## wherein R¹, R² and n are as defined as in claim1 and X is an activating group, and optionally off one or both of thefructosyl group or protective groups present and isolating the resultant3-(aminoacyl-amino)-saccharide.
 4. The process according to claim 3,wherein said resultant 3-(aminoacyl-amino)-saccharide is purifiedchromatographically.
 5. The process of claim 3, wherein the3-(aminoacyl-amino)-saccharide produced is selected from the group ofcompounds consisting of:3 -(4-L-aspartyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside, 3-(L-alanyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside, 3-(L-leucyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside,3-(L-lysyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside,3-(L-phenylalanyl-amino)-3-desoxy-D-allopyranosyl-β-D-fructofuranoside,and 3-(4-L-aspartyl-amino)-3-desoxy-D-allopyranose.
 6. The processaccording to claim 3, wherein said resultant3-(aminoacyl-amino)-saccharide is purified chromatographically.